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PubMed · 15537836

Combinatorial codon-based amino acid substitutions.

Jorge Yáñez·Martha Argüello·Joel Osuna·Xavier Soberón·Paul Gaytán
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Abstract

Twenty Fmoc-protected trinucleotide phosphoramidites representing a complete set of codons for the natural amino acids were chemically synthesized for the first time. A pool of these reagents was incorporated into oligonucleotides at substoichiometric levels to generate two libraries of variants that randomly carry either few or many codon replacements on a region encoding nine amino acids of the bacterial enzyme TEM-1 beta-lactamase. Assembly of the libraries was performed in a completely automated mode through a simple modification of ordinary protocols. This technology eliminates codon redundancy, stop codons and enables complete exploration of sequence space for single, double and triple mutations throughout a protein region spanning several residues. Sequence analysis of many non-selected clones revealed a good incorporation of the trinucleotides, producing combinations of mutations quite different from those obtained using conventional degenerate oligonucleotides. Ceftazidime-selection experiments yielded several never before reported variants containing novel amino acid combinations in the beta-lactamase omega loop region.

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Amino Acid SubstitutionCeftazidimeCodonCombinatorial Chemistry TechniquesGene LibraryMutagenesisOrganophosphorus Compoundsbeta-LactamasesJournal ArticleResearch Support, Non-U.S. Gov't

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Jorge Yáñez, Martha Argüello, Joel Osuna, Xavier Soberón, Paul Gaytán. 2004-11-10. Combinatorial codon-based amino acid substitutions.. https://doi.org/10.1093/nar%2Fgnh156

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